Carcinoma of the prostate is the most commonly diagnosed noncutaneous malignancy detected in men. In 2001 more than 198,000 American men were diagnosed with prostate cancer, and 31,500 died of this malignancy. Despite the curative intent of prostatectomy and radiotherapy, approximately 30% of early stage patients treated in such a fashion will relapse within 5-10 years of treatment. There is clearly a lack of fundamental knowledge of prostate cancer biology, and a lack of biomarkers that can accurately indicate prognosis or be used as targets for treatment. We propose to discover new markers through the use of the recently developed ICATTM technology. In the first step we will isolate membrane proteins from malignant tissues from a minimum of 4 prostate cancer patients and from tissues from a pool of 5 normal controls. In the second step, the proteins from the malignant tissues will be labeled with an isotopically heavy (d9) and the proteins from normal tissues with an isotopically light (d0) coded affinity tag (ICATTM), respectively. In the third step, the proteins of both samples will be combined and glycosylated proteins will be isolated, followed by trypsin digest. In the fourth step, this peptide mixture will be separated by multidimensional chromatography, and the fractions analyzed by automated tandem mass spectrometry (MS/MS). The raw data will be searched against protein and DNA databases and the differential peptide representation will be assessed using application specific software tools. These proteins will become candidate marker antigens which have potential as targets for diagnostic or therapeutic monoclonal antibodies.